Type: Oral
Session: 802. Chemical Biology and Experimental Therapeutics: Innovations in Therapy and Drug Screening
Hematology Disease Topics & Pathways:
Leukemia, Diseases, Non-Biological, Therapies, Biological Processes, Technology and Procedures, Lymphoid Malignancies, Myeloid Malignancies, proteomics
Our assay is based on a previously published “suicide gene”, a variant of the nucleotide salvage gene deoxycytidine kinase (DCK). This variant (DCK*) has mutations that increase its specificity for the synthetic substrate 2-Bromovinyldeoxyuridine (BVdU). Cells that express DCK* are killed by BVdU. We made mammalian expression vectors co-expressing GFP and DCK*, IKZF1, or a DCK*-IKZF1 fusion protein as a single bicistronic mRNA and stably introduced each into 293T cells. In the absence of POM, cells expressing DCK* or DCK*-IKZF1 are killed by BVdU . However, in the presence of an IKZF1 destabilizer, e.g. POM, the DCK*-IKZF1 protein is degraded, making the cells resistant to BVdU. Cells expressing DCK* alone are unaffected by POM and serve as a specificity control for the assay (Panel A).
We did a pilot chemical “up” screen with a ~ 2000 bioactive compound library (that included LEN and POM) using 293T cells expressing DCK*-IKZF1. In parallel, we conducted a “down” screen, using 293T cells co-expressing an IKZF1-Firefly Luciferase (Fluc) fusion protein and Renilla luciferase (Rluc) from a single bicistronic mRNA. Compounds that decreased the ratio of Fluc/Rluc activity were scored as hits. As expected, LEN and POM scored in both assays,but there was considerably more noise in the down assay. Lastly, we used the DCK*-IKZF1 cells to screen a library of uncharacterized IMiD derivatives. The screen correctly identified a novel IMiD derivative (MI-2-61) and a known next-generation IMiD (Avadomide) with greater potency against IKZF1 than POM.
To identify novel degraders of IKZF1, we used the DCK*-IKZF1 cells and DCK* control cells to screena metabolic inhibitor/anticancer library of ~600 compounds. We identified Spautin-1 as a compound that rescues DCK*-IKZF1 cells, but not DCK* control cells from BVdU toxicity. Spautin-1 downregulates exogenous IKZF1 in 293T cells (Panels B and C) and endogenous IKZF1 in KMS11 myeloma cells. Spautin-1 reportedly suppresses autophagy through inhibition of USP10 and USP13. However, siRNA mediated knockdown of USP10 and USP10 neither altered IKZF1 protein levels, nor blocked downregulation of IKZF1 by Spautin-1. Moreover, Spautin-1 downregulated IKZF1 in 293FT cells in which autophagy was disabled by CRISPR/Cas9-mediated disruption of genes crucial to the autophagy pathway suggesting that the downregulation of IKZF1 by Spautin-1 occurs via a novel mechanism.
Unlike IMiDs, the downregulation of IKZF1 by Spautin-1 does not require the CRBN E3 ligase. However, it is blocked by inhibitors of the E1 ubiquitin activating enzyme or the proteasome, but not by neddylation inhibitors required for Cullin-dependent E3 ligases. These data suggest that Spautin-1 triggers the proteasomal degradation of IKZF1 using a non-Cullen E3 ligase. The downregulation of exogenous IKZF1 by Spautin-1 requires the N-terminus of IKZF1, but not the zinc finger domain (ZF2) targeted by IMiDs. Preliminary structure-activity relationship (SAR) studies identified both active and inactive Spautin-1 derivatives, suggesting that downregulation of IKZF1 by Spautin-1 reflects a specific protein binding event and that Spautin-1’s potency and specificity can be optimized further.
We are undertaking studies to identify the mechanism by which Spautin-1 degrades IKZF1 in the hope that it may represent a novel druggable pathway to therapeutically degrade IKZF1, and validate the use of this platform to discover degraders of “undruggable” targets in other cancers.
Disclosures: Koduri: Cedilla Therapeutics: Consultancy. Paulk: Novartis: Current Employment. Harris: ONO Pharmaceutical: Consultancy. Buhrlage: Adenoid Cystic Carcinoma Foundation: Other: Scientific Advisory Board. Kaelin: Cedilla Therapeutics: Other: Scientific Founder; Eli Lilly: Membership on an entity's Board of Directors or advisory committees; Fibrogen: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Tango Therapeutics: Other: Founder; Tracon Therapeutics: Membership on an entity's Board of Directors or advisory committees.